PROJECT SUMMARY Our long-term goal is to elucidate the molecular mechanisms that restructure the genome to allow for the transition from a specified cell type to a pluripotent state. During the initial stages of embryonic development, the genomes of the specified germ cells are rapidly and efficiently reprogrammed to generate the pluripotent cells of the early embryo. Many features are shared between the efficient reprogramming that occurs in the early embryo and the much less efficient reprogramming that occurs in culture. The capacity to generate pluripotent stem cells has great potential in the modelling and treatment of disease. Understanding the fundamental molecular mechanisms that drive reprogramming will have important implications in our ability to rapidly and reproducibly induce the pluripotent state. Both in culture and in the early embryo, reprogramming requires the activity of specialized transcription factors, termed pioneer factors. Pioneer factors are distinctive in that, unlike other transcription factors, they can bind to DNA in the context of nucleosomes. This feature allows them unique access to the genome and helps to redefine the chromatin accessibility landscape. Nonetheless, it remains unclear what specific functions of pioneer factors are required to drive reprogramming and what the barriers are to pioneer factor-mediated reprogramming. In Drosophila the transcription factor Zelda is required for the initial transcriptional activation of the zygotic genome. We have demonstrated that Zelda possesses essential features of pioneer transcription factors and that this activity is required throughout the process of zygotic genome activation. However, Zelda is unlikely to be working independently, and we have implicated another pioneering factor, GAGA factor, as functioning together with Zelda to define accessible chromatin domains in the early embryo. By combining our development of novel tools to interrogate transcription factor function in the early embryo with the strengths of the well-studied fly system, we are uniquely positioned to determine essential features of reprogramming. We will use genetic, genomic, biochemical, and imaging strategies to 1) define how pioneering factors cooperate to reprogram the zygotic genome, 2) identify chromatin barriers to pioneer factor- mediated reprogramming and 3) determine the role of intrinsically disordered regions in establishing chromatin domains in the early embryo. Our proposed research is significant because by defining essential characteristics of pioneering transcription factors we will identify unifying principles required for efficient reprogramming of specified cells to pluripotency.